CASE REPORTpublished: 05 June 2018

doi: 10.3389/fped.2018.00163

Frontiers in Pediatrics | www.frontiersin.org 1 June 2018 | Volume 6 | Article 163

Edited by:

Enrico Baruffini,

Università degli Studi di Parma, Italy

Reviewed by:

Muhammad Jawad Hassan,

National University of Sciences and

Technology, Pakistan

Beate Peter,

University of Washington Tacoma,

United States

*Correspondence:

Antonio Benítez-Burraco

abenitez8@us.es

Specialty section:

This article was submitted to

Genetic Disorders,

a section of the journal

Frontiers in Pediatrics

Received: 12 February 2018

Accepted: 15 May 2018

Published: 05 June 2018

Citation:

Benítez-Burraco A,

Barcos-Martínez M, Espejo-Portero I,

Fernández-Urquiza M, Torres-Ruiz R,

Rodríguez-Perales S and

Jiménez-Romero MS (2018)

Narrowing the Genetic Causes of

Language Dysfunction in the 1q21.1

Microduplication Syndrome.

Front. Pediatr. 6:163.

doi: 10.3389/fped.2018.00163

Narrowing the Genetic Causes ofLanguage Dysfunction in the 1q21.1Microduplication SyndromeAntonio Benítez-Burraco 1*, Montserrat Barcos-Martínez 2,3, Isabel Espejo-Portero 2,3,

Maite Fernández-Urquiza 4, Raúl Torres-Ruiz 5, Sandra Rodríguez-Perales 5 and

Ma Salud Jiménez-Romero 6

1Department of Spanish, Linguistics, and Theory of Literature, University of Seville, Seville, Spain, 2 Laboratory of Molecular

Genetics, University Hospital “Reina Sofía”, Córdoba, Spain, 3Maimónides Institute of Biomedical Research, Córdoba, Spain,4Department of Spanish Philology, University of Oviedo, Oviedo, Spain, 5Molecular Cytogenetics Group, Centro Nacional

Investigaciones Oncológicas, Madrid, Spain, 6Department of Psychology, University of Córdoba, Córdoba, Spain

The chromosome 1q21.1 duplication syndrome (OMIM# 612475) is characterized by

head anomalies, mild facial dysmorphisms, and cognitive problems, including autistic

features, mental retardation, developmental delay, and learning disabilities. Speech and

language development are sometimes impaired, but no detailed characterization of

language problems in this condition has been provided to date. We report in detail

on the cognitive and language phenotype of a child who presents with a duplication

in 1q21.1 (arr[hg19] 1q21.1q21.2(145,764,455-147,824,207)×3), and who exhibits

cognitive delay and behavioral disturbances. Language is significantly perturbed, being

the expressive domain the most impaired area (with significant dysphemic features in

absence of pure motor speech deficits), although language comprehension and use

(pragmatics) are also affected. Among the genes found duplicated in the child, CDH1L is

upregulated in the blood of the proband. ROBO1, a candidate for dyslexia, is also highly

upregulated, whereas, TLE3, a target of FOXP2, is significantly downregulated. These

changes might explain language, and particularly speech dysfunction in the proband.

Keywords: chromosome 1q21.1 duplication syndrome, cognitive delay, language deficits, speech problems,

CDH1L, ROBO1

INTRODUCTION

Copy number variant (CNV) syndromes entailing language impairment provide importantevidence of how changes in gene dosage impact on the wiring and function of brain areasinvolved in language processing. The 1q21.1 region contains several segmental-duplication blockswhich favor the occurrence of microdeletions and microduplications. The chromosome 1q21.1duplication syndrome (OMIM# 612475) results from microduplications of the BP3-BP4 region,with a minimum duplicated region of ∼1.2Mb of unique DNA sequence, which includes at leastseven genes, although a less common variant, resulting from microduplications of the BP2-BP4region, with a duplicated region of ∼2Mb and encompassing the region deleted in the TARsyndrome (OMIM# 274000), has been also reported (1). These two variants are commonly referredas Class I 1q21.1 duplication syndrome and Class II 1q21.1 duplication syndrome, respectively.

Benítez-Burraco et al. Language Dysfunction and 1q21.1 Microduplication

Many of the subjects bearing the 1q21.1 microduplicationexhibit autistic features, attention deficit-hyperactivity disorder,mild or moderate mental retardation, relative macrocephaly,mild dysmorphic features, heart disease, and hypotonia(1–3). Some of these features are shared with carriers ofthe reciprocal deletion, including cognitive dysfunction,motor problems, articulation deficits, and hypotonia (4).Interestingly, microcephaly is prevalent in deletion carriers,whereas macrocephaly is common in duplication carriers;likewise, deletions are usually associated with schizophrenia,while carriers of the duplication usually exhibit autisticfeatures [(4) and references herein]. Interestingly too, the adultphenotype seems to be milder than the child presentation of thesyndrome and includes macrocephaly and abnormalitiesof possible connective tissue origin, and occasionally,schizophrenia (3). Overall, because the microduplication(as well as the reciprocal deletion) is associated with a highlyvariable phenotype, and because it exhibits an incompletepenetrance and variable expressivity, it has been suggestedthat this is a susceptibility locus, rather than a clinicallydistinct syndrome, which predisposes to neurodevelopmentalproblems (5).

Little is known about the genetic causes of theneuropsychiatric phenotype of 1q21.1 duplications. Thedistinctive genomic architecture of the 1q21.1 region, whichaccounts for its genomic instability, seems to be evolutionarilyadvantageous, and includes multiple human-specific changes(compared to extant primates) important for brain evolution,such as the expansion of genes coding for proteins withDUF1220 protein motifs, and the duplicative transpositions ofnon-1q21.1 genes, like SRGAP2 and HYDIN (6). According toDolcetti et al. (3), gene expression data suggest that candidategenes for the distinctive cognitive profile of people with the1q21.1 microduplication may include BCL9, GJA8, PDZK1,and PRKAB2. Nonetheless, no conclusive genotype-phenotypecorrelations have been found. This is particularly true of thelanguage problems that are commonly observed in patients,among other reasons, because we still lack a comprehensivecharacterization of the language phenotype associated to thiscondition.

In this paper, we report on a boy with a microduplicationof the 1q21.1 region and provide a detailed description of hislanguage (dis)abilities. Relying on our current knowledge of thegenetic aspects of language development and language evolution,but also on ad-hoc analyses of the expression pattern of selectedgenes in the blood of our proband, we propose BCL9 andCDH1L as principal candidates for language dysfunction in thissyndrome.

MATERIAL AND METHODS

Linguistic, Cognitive, and behavioralAssessmentThe global developmental profile of the proband was evaluatedwith the Spanish versions of the Battelle DevelopmentalInventories (7), the Luria-Nebraska Neuropsychological Battery

for Children (LNNB-C) (8), the Kaufman Assessment Battery forChildren (K-ABC) (9), and the Inventory for Client andAgency Planning (ICAP) (10). The Spanish version of theAutism Diagnostic Observation Schedule- Revised (ADOS-R)was used, specifically, for screening autistic features in thechild.

Battelle Developmental InventoriesBattelle Developmental Inventories consist of 341 items andare aimed to evaluate personal/social development, adaptivecapabilities, motor abilities (gross and fine), communicationskills (in the receptive and expressive domains), and cognitivedevelopment.

Luria-Nebraska Neuropsychological Battery for

Children (LNNB-C)The LNNB-C is aimed to evaluate (and anticipate) the child’slearning, experience, and cognitive skills, as well as hisneuropsychological deficiencies. The test comprises 10 scales thatare aimed to measure executive functioning in several areas ofinterest (manual mobility, left-right orientation, gestures andpraxias, verbal regulation, spatial orientation) as well as languagefunctioning in different domains (object and drawing naming,phonological awareness, vocabulary in pictures, similarities anddissimilarities, and numerical operations).

Kaufman Assessment Battery for Children (K-ABC)The K-ABC is aimed to evaluate diverse processing and cognitiveabilities as they are put into use, either simultaneously orsequentially, to resolve specific problems. The test comprises16 subtests arranged into three scales: Simultaneous Processing,Sequential Processing, and Knowledge.

Inventory for Client and Agency Planning (ICAP)The ICAP is aimed to evaluate the subject’s functional abilitiesand maladaptive behaviors in the following general areas:motor skills, social and communication skills, personal livingskills, and community living skills. The ICAP measures thefrequency and severity of eight types of behavioral disturbances,which are organized in three subscales: asocial maladaptivebehavior (uncooperative behavior and socially offensivebehavior), internalized maladaptive behavior (withdrawn orinattentive behavior, unusual or repetitive habits, and self-harm),and externalized maladaptive behavior (disruptive behavior,destructive to property, and hurtful to others). Behavior israted as normal or abnormal, whereas behavioral problems aresubsequently rated as marginally serious, moderately serious,serious, or very serious.

Autism Diagnostic Observation Schedule-Revised

(ADOS-R)This test is designed for toddlers between 18 and 60 monthsand comprises 23 questions. A positive score is indicative of thepossibility of suffering from the disease.

Regarding the language profile of the child, his abilities wereevaluated in detail with the Prueba de Lenguaje Oral de Navarra-Revisada [Navarra Oral Language Test, Revised] (PLON-R) (11),

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and the Spanish version of the Peabody Picture Vocabulary Test,Third Edition [PPVT-3; (12)].

Prueba de Lenguaje Oral de Navarra-Revisada

(PLON-R)This test was used to assess in depth the child’s expressive abilities.The PLON-R is aimed for children between 3 and 6 years,and characterizes language production both structurally andfunctionally. The composite score “form” evaluates speech soundproduction at the level of single word, phonological awareness,as well as morphological and syntactic features of the child’simitated and elicited verbal production. The composite score“contents” assesses word and sentence meanings. Finally, thetest also evaluates different aspects of language use (planning,autoregulation, and modification under environmental cues) insemi-spontaneous conditions.

Peabody Picture Vocabulary Test (PPVT-3)This test was used to evaluate in more detail the receptive abilitiesof the proband. The PPVT-3 assesses the correct acquisition ofwords by the child via several tasks in which the child is asked topoint to one of four color pictures on a page after hearing a word.

Additionally, language in use and overall communicationskills in spontaneous conditions were assessed through thesystematic analysis of a 17-min sample of the child’s talkin interaction with his mother. The conversation was video-recorded and then transcribed and coded using CHAT (Codesfor the Human Analysis of Transcripts) software, a toolof the CHILDES Project (13). CHAT allows to code forspeech production phenomena (including information aboutarticulation, prosody, and fluency) in the main lines of thetranscript, and for phonological, morphological, syntactic andpragmatic phenomena in dependent lines.

It has to be pointed out that, when evaluating languagein use, every linguistic phenomenon is to be assessed from apragmatic perspective (14). Thus, we first tagged the linguisticerrors made by the child indicating the linguistic componentsaffected (phonology, morphology, syntax, lexical semantics).Then we evaluated the communicative effects of each errorand labelled it accordingly on the pragmatic level, using thePragmatic Evaluation Protocol for the analysis of oral Corpora(PREP-CORP), which has been satisfactorily used to providethe linguistic profile of other neurogenic disorders (15–17).The number of occurrences of each labeled phenomenon wasautomatically computed by means of CLAN (ComputerizedLanguage Analysis) (18), allowing us to build up a globaldescription of the pragmatic linguistic profile of the proband, thatis to say, of the most salient phenomena of his language in use.

Finally, the linguistic profile of the proband’s parents wasassessed with the verbal scale of the Spanish version of theWAIS-III (19), the Test de Aptitud Verbal “Buenos Aires” [BuenosAires Verbal Aptitude Test] (BAIRES) (20), the Batería para laEvaluación de los Procesos Lectores en Secundaria y Bachillerato[Battery for the evaluation of reading abilities in high schoolstudents] (PROLEC-SE) (21), and one specific task aimed toevaluate the comprehension of passives and correferences.

WAIS-IIIThe verbal scale of the WAIS-III comprises six tasks aimed toassess the subject’s abilities in two different domains of languageprocessing: verbal comprehension [Similarities (S), Vocabulary(V), Information (I), and Comprehension (CO)] and workingmemory [Digits (D) and Arithmetic (A)].

BAIRESThe BAIRES is aimed to evaluate language comprehension andproduction in subjects above 16 years old. It comprises two taskswhich assesses the subject’s ability to understand and providewith synonymic words and verbal definitions, respectively.

PROLEC-SEThe PROLEC-SE is aimed to assess the reading abilities byadolescents between 12 and 18 years old. It comprises six taskswhich evaluate three different processes involved in reading:lexical (word reading, pseudoword reading), semantic (textcomprehension, text structure), and syntactic (picture-sentencemapping, punctuation marks).

Ad-hoc taskThe ad-hoc task was a sentence-picture-matching task aimedto evaluate the comprehension of bound anaphora in complexsentences (that is, the ability to properly bind a determiner phrasein the subordinated clause to a referential element in the mainclause) and of canonical long passive structures (that is, with a by-phrase). While the former task enquires about highly-demandingcomputational abilities in the domain of language processing, thelatter is usually difficult for people with poor reading abilities,because in Spanish passives are much more frequent in theliterary register.

Molecular and cytogenetic AnalysisDNA from the patient and his parents was extracted from 100 µlof EDTA-anticoagulated whole blood using MagNA Pure (RocheDiagnostics, West Sussex, UK) and used for subsequent analyses.

Karyotype AnalysisPeripheral venous blood lymphocytes were grown followingstandard protocols and collected after 72 h. A moderateresolution G-banding (550 bands) karyotyping by trypsin (Gibco1x trypsin R© and Leishmann stain) was subsequently performed.Microscopic analysis was performed with a Nikon R© eclipse50i optical microscope and the IKAROS Karyotyping System(MetaSystem R© software).

Fragile X Syndrome AnalysisCGG expansions affecting the gene FMR1 (the main determinantfor Fragile X syndrome) were analyzed in the patient accordingto standard protocols. Polymerase chain reaction (PCR) of thefragile site was performed with specific primers for the fragileregion of the FMR1 locus and the trinucleotide repeat size of theresulting fragments was evaluated by electrophoresis in agarosegel.

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Multiplex Ligation-Dependent Probe Amplification

(MLPA)MLPA was performed to detect abnormal CNV of thesubtelomeric regions of the probands’ chromosomes. MLPA isbased on the amplification (by use of a single PCR primerpair) of many different probes, each of which detecting aspecific subtelomeric DNA sequence. Three different kits fromMRC-Holland were used: SALSA R© MLPA R© probemix P036-E3SUBTELOMERES MIX 1, SALSA R© MLPA R© probemix P070-B3 SUBTELOMERES MIX 2B, and SALSA R© MLPA R© probemixP245-B1 Microdeletion Syndromes-1A. The PCR products wereanalyzed by capillary electrophoresis in an automatic sequencerHitachi 3500 and further analyzed with the Coffalyser V 1.0software from MRC-Holland.

Microrrays for CNVs Search and Chromosome

Aberrations AnalysisThe DNA from the patient and his parents was hybridized ona CGH platform (Agilent Technologies). The derivative log ratiospread (DLRS) value was 0.172204. The platform included 60.000probes. Data were analyzed with Agilent CytoGenomics 3.0.5.1and qGenViewer, and the ADM-2 algorithm (threshold= 6.0; abs(log2ratio)= 0.25; aberrant regions had more than 4 consecutiveprobes).

qPCRThe expression pattern of genes of interest was assessed in bloodby qRT-PCR in order to determine possible differences betweenthe proband and his parents. Total RNA extraction and qRT-PCR were done as previously described (22). Briefly, total RNAwas extracted from cells using Trizol (Sigma Aldrich) accordingto the manufacturer’s procedure, followed by a treatment withRNase-free DNase (Roche). cDNA was synthesized from 1 µg oftotal RNA using a Superscript III First Strand cDNA synthesis kit(LifeTechnologies). Amounts of specific mRNAs in samples werequantified by qRT-PCR using an ABIPrism 7900 HT DetectionSystem (Applied Biosystems) and SYBR green detection. PCRwas performed in 96-well microtest plates (Applied Biosystems)with 0.5 units of Taq Polymerase (Applied Biosystems) per welland 35–40 cycles. In all experiments, mRNA amounts werenormalized to the total amount of cDNA by using amplificationsignals for hGUSB. Each sample was determined in triplicate,and at least three independent samples of each patient wereanalyzed. Primer sequences and PCR conditions are listed inSupplementary Table 1.

Ethics approval for this research was granted by the ComitéÉtico del Hospital “Reina Sofía.” Written informed consent wasobtained from the proband’s parents for publication of this casereport and of any accompanying tables and images.

RESULTS

Clinical HistoryThe proband (Figure 1A) is a boy born by normal deliveryafter 37 weeks of risk gestation because of placenta previa withbleeding and threatened abortion. The mother was a 28-year-old woman, who suffered from high blood pressure and diabetes

mellitus during pregnancy and who consumed anxiolytic drugsduring the first trimester. At the delivery, no signs of diseasewere observed in the newborn. The child suffered from recurrentinfantile colic during the first months of life, as well as fromirritability that seriously disturbed his sleeping. At age 2 yearsand 3 months, his weight was 9.490 kg (percentile 1), his height86.5 cm (percentile 18) and the occipitofrontal circumference41.2 cm (below percentile 1). At present, when he is 6 yearsold, his weight is 19 kg (percentile 21), his height 117 cm(percentile 62) and the occipitofrontal circumference 50 cm(around percentile 30). Cranial magnetic resonance imaging(MRI), performed at 5 years and 7 months of age, yieldednormal results. The child was set twice a tympanic drainage inconjunction with adenoidectomy (at 3 years and 4 years). At thattime, the Eustachian tube was reported to be underdeveloped,although no auditory impairment was observed. One paternalcousin of the proband is dyslexic, whereas a second paternalcousin (from a different brother of the father) suffers fromlanguage developmental delay. Finally, the child exhibits facialdysmorphisms, including mild hypertelorism, small nose, andprotruding ears.

Language and Cognitive DevelopmentDevelopmental anomalies were first reported by the teachers ofthe nursery school when the proband was 2 years old. The childwas unable to stand up without aid and fine and gross motorabilities seemed severely delayed. Bladder and bowel control hadnot been achieved either. The child only babbled and no singleword was observed. Stereotypic behavior was reported. The childreceived early childhood intervention from age 2 years and 4months. At age 2 years and 8 months his global developmentwas assessed in detail with the Spanish version of the BattelleDevelopmental Inventories. The resulting scores were suggestiveof a mild developmental delay, mostly impacting on his language,social and adaptive abilities, whereas his cognitive skills wereslightly above the mean (Figure 1B). When he was 3 years old,the boy started attending a normal nursery school.

At age 5, the parents reported a generalized cognitive andbehavioral regression. Stereotypic behaviors increased and thechild exhibited lack of cognitive flexibility, occasional spatialdisorientation, obsessive behavior, perseverations, unmotivatedfears to darkness or load sounds, and lack of interaction withhis peers at school. Although he seemed to have mastered a goodvocabulary, he failed to put it into use for normal interaction. Healso had problems with the conceptualization of time. Moreover,although he received speech and cognitive aid and therapy on adaily basis, at school he failed in achieving the learning objectivesplanned for that age.

At age 5 years and 4 months, the Spanish version of theBattelle Developmental Inventories was administered again.The obtained scores were not suggestive of a generalizedregression, although the child’s cognitive skills seemed moreimpaired than at younger ages (Figure 1B). The Spanishversion of the Kaufman Assessment Battery for Children wasalso administered to evaluate his learning abilities. The mostimpaired areas were the general mental processing abilities(very significant deficit) and the sequential processing abilities

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FIGURE 1 | Main physical and cognitive findings in the proband. (A) Facial picture illustrating some of the dysmorphological features found in the proband. Written

informed consent was obtained from the parents of the child for the publication of this image. (B) Developmental profiles of the proband at 2 years and 8 months (blue)

and at 5 years and 4 months (orange) according to the Battelle Developmental Inventories. In order to make more reliable comparisons, the resulting scores are shown

as relative values referred to the expected scores according to the chronological age of the child. DA, developmental age; CA, chronological age. (C) Developmental

profile of the proband at age 5 years and 6 months according to Luria-Nebraska Neuropsychological Battery for Children (LNNB-C). DA, developmental age; CA,

chronological age. (D) Developmental profile of the proband at age 5 years and 6 months according to the Inventory for Client and Agency Planning (ICAP).

(significant deficit), although the child scored lower than hispeers in the remaining domains too. These results reinforce theview that the widespread regression reported by the parentswas not for real, and that the child’s poor performance indaily tasks, which becomes more evident as the boy growsolder, might result from a moderate learning deficit causedby his linguistic and cognitive deficits. Additionally, in viewof the behavioral problems reported by parents and teachers,but also of the scores obtained in the Batelle DevelopmentalInventories, the Spanish version of the Autism DiagnosticObservation Schedule- Revised (ADOS-R) was administered atthe age of 5 years and 5 months. The child did not fulfillthe requirements for Autism Spectrum Disorders (ASDs). Hescored normally in all the tasks, including the use of non-verbal aspects of social interaction, interactive symbolic play,reciprocity during interaction and play, and understanding of,and response to, others’ beliefs. The boy usually paid attentionto others and was able to express his own feelings toward themand about himself. What is more, the qualitative analysis ofthe interactive aspects of the spontaneous conversation withhis mother confirmed the absence of autism. In truth, theautistic-like behaviors initially reported by parents and teachers(avoidance behaviors, obsessive behaviors, lack of visual contact)were mostly observed when the child was placed in an unfamiliarenvironment and felt stressed, unsecure, or anxious. It is alsopossible that he suffered from some sort of compulsive-obsessivedisorder.

When the child was 5 years and 6 months old, his globallinguistic and cognitive profile, as well as his learning abilities

were also evaluated with the Luria-Nebraska NeuropsychologicalBattery for Children (LNNB-C). The obtained scores weresuggestive of a deep impairment of language and executivefunctions (in all the assessed domains the scores corresponded topercentiles 1–2) (Figure 1C). Likewise, the Inventory for Clientand Agency Planning (ICAP) was administered for assessingthe child’s adaptive behavior. The obtained scores (Figure 1D)were indicative of a delay of nearly 2 years in his globaldevelopment, being social and communication skills the mostimpaired domain.

Because of the significant impairment and delay observed inthe language domain, we also evaluated in detail the linguisticabilities of the proband at the age of 5 years and 6 months. Theglobal score obtained in the Prueba de Lenguaje Oral de Navarra-Revisada (PLON-R), which evaluates speech sound production atthe single word level as well as expressive abilities, was suggestiveof a mild delay in the acquisition of the expressive componentof language, being phonological awareness and morpho-syntaxthe most impaired domains. Likewise, a mild delay was suggestedregarding the use of language. On the contrary, the childscored like their peers regarding the meaning of words andsentences. Concerning his receptive abilities in the domain oflanguage, the global score obtained in the Spanish version ofthe Peabody Picture Vocabulary Test (PPVT-3) was suggestiveof a delay of 12 months in the acquisition of the meaning ofwords, with an expected negative impact of his comprehensionabilities.

Lastly, we examined in detail the child’s language in useand his communication skills through the analysis of a

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naturally-occurring interaction with his mother. The proband’sspeech production abilities in natural settings were severelyimpaired, hindering the child’s intelligibility and communicationsuccess. Fluency problems included syllabified and effortfularticulation, reduplication of syllables in any position of theword, and severe alterations of rhythm, use of pauses, volume,and prosody. Assimilation of specific sounds was frequentin the boy’s speech, as well as simplifications of consonantclusters and deletions of weak syllables. Omissions of strongsyllables (those bearing the primary stress) and of sounds ininitial position were occasionally observed too. Concerninglanguage structure and complexity, the child’s mean length ofutterance in words (MLUw) was 2.6, which is within the rangeobserved in children aged between 2 years and 6 months,and 2 years and 11 months (23), and thus remarkably lowerthan the expected value by age. In longer utterances, thechild produced morpho-syntactic errors, such as additions orsubstitutions of prepositions, or omissions of weak pronouns(usually, personal pronouns functioning as the object of thesentence [me “me” vs. yo “I”]), which may be due to thewidespread weak-syllable deletions observed in his discourse.Finally, his interactive pragmatic skills seemed to be preserved:visual contact and joint attention mechanisms were normal, aswell as patterns of turn taking and construction of collaborativeturns during conversation. However, enunciative pragmaticskills (that is, principles of language use regulating howutterances are interpreted in their contexts of usage), such asthe observation of maxims of quality (“be truthful and donot give information that is false or that is not supported byevidence”) and relation (“be relevant, and say things that arepertinent to the discussion”) (24), were slightly impaired, whichmight be related to his attention problems and/or intellectualdisability.

Cytogenetic and Molecular AnalysesRoutine cytogenetic and molecular analyses of the proband wereperformedwhen hewas 2 years and 7months old. PCR analysis ofthe FMR1 fragile site was normal. MLPA of subtelomeric regionswas normal too. A comparative genomic hybridization array(array-CGH) identified a duplication of 2.1Mb in the 1q21.1region (arr[hg19] 1q21.1-q21.2(145,764,455-147,824,207)×3)(Figure 2A). The duplicated region corresponds to the regionduplicated in Class II 1q21.1 duplication syndrome and includes38 genes of which 13 are protein-coding genes (Figure 2B).

As noted, our proband exhibits most of the physical,behavioral, and cognitive features of the 1q21.1 microduplicationsyndrome (Table 1). The duplication was inherited from theproband’s non-symptomatic mother (Figure 3A). The womanexhibited a verbal IQ slightly above the mean, although withpeaks and valleys, which are suggestive of relative strengths inthe domain of attention and auditory memory (task Digits)and of relative weaknesses in the domain of symbol processing(task Arithmetic) (Figure 3B). Additionally, she exhibited avery good command of verbal abilities as assessed by theBAIRES test, around percentile 90th, and normal-to-high readingabilities (Figure 3C). We have not assessed other features of themicroduplication. The fact that she suffered from anxiety during

pregnancy might be indicative of some affectedness in otherdomains [see (4), Table 2], but as far as language and cognitionare concerned, she performed normally.

In order to delve into the molecular causes of the speechand language problems exhibited by the child, we surveyed theliterature looking for other clinical cases in which languagedeficits have been linked or associated to the mutation or thedysfunction of any of the genes duplicated in our proband. Wefound that CHD1L is a candidate for autism spectrum disorder(ASD) and attention deficit hyperactivity disorder (ADHD)(25, 26), whereas PRKAB2 and BCL9 have been associated toschizophrenia (27–30).

We also relied on DECIPHER (https://decipher.sanger.ac.uk/) to find cases of CNVs affecting chromosomal fragmentssmaller than the region duplicated in the child, that may helpassociate language dysfunctions to specific genes. We found thatpatients bearing microduplications not encompassing the genesCHD1L and BCL9 do not usually exhibit language problems.Conversely, duplications involving CHD1L entail delayed speechand language development. No patient bearing a duplicationaffecting the BCL9 only has been found (Figure 4).

Finally, we used String 10.5 (www.string-db.org) forexamining potential functional links between the genesduplicated in our proband and genes important for language,in particular, known candidate genes for language disorders[dyslexia and SLI, as compiled by (31–33)] and for languageevolution, as posited by Boeckx and Benítez-Burraco(34, 35) [importantly, many of these genes are also relatedto language dysfunction in broader cognitive disorders,particularly, autism and schizophrenia, as discussed in (36–38)](Supplementary Table 2). String 10.5 predicts physical andfunctional associations between proteins relying on differentsources (genomic context, high-throughput experiments,conserved coexpression, and text mining) (39). Several proteinswere predicted to be direct interactors of some of the genes foundin the duplicated fragment: FOXO1, TP53, TSC1, for PRKAB2;PARP1, for CHD1L, and CTNNB1, ARX, TL3, and TL2, forBCL9 (Figure 5).

In order to check the biological reliability of these potentiallinks and the possibility that other robust candidates for languagedysfunction were affected in our proband, we performed qRT-PCR analyses of selected genes using blood from the proband andhis parents. We determined the expression level of (i) protein-coding genes located within the duplicated fragment (BCL9,GJA8, GJA5, ACP6, PDZK1, PRKAB2, CHD1L, HYDIN2), (ii)functional partners of the protein-coding genes located withinthe duplicated fragment with a known role in cognition/languagedevelopment and/or impairment, as found in the literature andas predicted by String 10.5 (PARP1, CTNNB1, ARX, FOXP2,TLE2, TLE3, FOXO1, TSC1), and (iii) other strong candidatesfor language disorders and language evolution, as discussed inBoeckx and Benítez-Burraco (34, 35) (AUTS2, BAZ1B, BMP2,CNTNAP2, DCDC2, DLX1, ELP4, FOXP1, GRIN2A, POU3F2,ROBO1, RUNX2, SLIT2, and SOX9) (Figure 4). We found that4 genes are significantly upregulated or downregulated in theproband compared to his healthy parents: CHD1L, ACP6, TL3,and ROBO1 (Figure 6 and Supplementary Table 3).

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FIGURE 2 | Chromosomal alterations found in our proband. (A) Screen capture of the array-CGH of the proband’s chromosome 1 showing the microduplication at

1q21.1. (B) Screen capture of the UCSC Genome Browser (https://genome.ucsc.edu/) showing the genes duplicated in the proband.

DISCUSSION

The widespread application of next generation sequencingfacilities to the genetic diagnosis of people with cognitiveand language disorders has resulted in a growing number ofgenes and chromosomal regions associated to these conditions.Nonetheless, in most cases, no robust genotype-to-phenotype

correlations have been found. In this paper, we have characterizedthe cognitive profile of a boy bearing a microduplication in1q21.1, with a focus on his language deficits. The boy exhibitsmany of the cognitive, behavioral, and physical symptoms ofthe 1q21.1 microduplication syndrome (Table 1). Regarding thelanguage (dis)abilities of the affected people, the most detailedaccount is Bernier et al. (4), who found that ∼10% of their

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TABLE 1 | Summary table with the most relevant clinical features of our proband

compared to other patients with the 1q21.1 microduplication syndrome [source:

Unique (https://www.rarechromo.org/), (1, 2, 4)].

Clinical features

COGNITIVE/BEHAVIORAL FEATURES

Autism or autistic behaviors

Anxiety and mood disorders x

Attention deficit-hyperactivity disorder (ADHD)

Mild-to-moderate mental retardation x

Mild-to-moderate developmental delay x

Learning disabilities x

Language disorder x

NEUROLOGICAL FINDINGS

Fine motor impairment x

Coordination disorder x

Hypotonia x

Articulatory problems/stuttering x

Seizures

Sleep disturbances x

Agility abnormalities x

PHYSICAL FEATURES

Macrocephaly (occasional craniosynostosis)

(Mild) dysmorphic facial features

Frontal bossing

Wider space between the eyebrows and above the nose

Mild hypertelorism x

Small nose x

Prominent epicanthic folds

Wide, flat nasal bridge

Low-set ears x

Stature below the average

Scoliosis

OTHER MEDICAL PROBLEMS

Gastric ulcers

Heart disease

Minor genital anomalies (hypospadias, testicles undescended at birth)

subjects suffered from language disorder, whereas phonologicalprocessing disorder was observed in ∼ 5% of cases. Likewise,around 10% of DECIPHER patients with a duplication within,overlapping, or encompassing the region duplicated in ourproband (N = 224) are reported to suffer from languageproblems. Nonetheless, the linguistic evaluation of patientsusually involves psychological tests only. To the best of ourknowledge, this is the first time a detailed linguistic profile of asubject with the 1q21.1 microduplication has been provided.

Accordingly, we have conducted a fine-grained analysis ofthe language strengths and weaknesses of our subject with the1q21.1 microduplication syndrome using a battery of specificdiagnostic tools, as well as examining his language performancein spontaneous conditions, both at the structural and thefunctional levels. Overall, we found conclusive evidence of aglobal delay, which plausibly results from delayed and atypical

language development in the expressive and receptive domains.The proband’s language impairment would be on the basis ofa learning disability that precludes the normal acquisition ofexecutive functions and adaptive behavior, hindering the child’sinteraction with his social environment.

The expressive problems exhibited by the child seem to bealso due to altered speech processes, in the form of dysphemia(frequent repetitions or prolongations of sounds and syllables,as well as frequent pauses interrupting the flow of the discourse)and dysprosody (abnormal presentation of prosodic features, likestress, intonation, melody, or rhythmic patterns), as could beobserved during the natural spontaneous interaction between thechild and his mother. These problems might result in part fromdeficient executive functions, because we have found no evidenceof pure motor speech disorders, like apraxia or dysarthria.Finally, our proband seemingly suffers from a mild pragmaticdeficit, because of the also attested delay in the masteringof conversational maxims [see (40, 41) for the neurotypicalpopulation].

Regarding the molecular causes of the observed symptoms,Dolcetti et al. (3) point out that UCSC gene expression data aresuggestive of the involvement of the genes BCL9, GJA8, PDZK1,and PRKAB2 in the neuropsychiatric phenotype of the 1q21.1duplication syndrome. O’Bleness et al. (6) have pointed out thatthe region found duplicated in our proband contains severalcandidates for human-lineage-specific neurodevelopmentalchanges, including most of the genes encoding DUF1220protein domains (NBPF10, NBPF11, NBPF12, NBPF24), aswell as PDZK1 and HYDIN2. Among the genes located withinthe duplicated fragment, we have found that 2 genes havechanged their expression level in the blood of the affected subjectcompared to his healthy father and his asymptomatic carriermother. Hence, ACP6 is significantly dowregulated, whereasCHD1L is significantly upregulated (Figure 6).

ACP6 encodes a histidine acid phosphatase involved inG protein-coupled receptor signaling and the balancing oflipid composition within the cell. Interestingly, differencesin the methylation status of the gene have been correlatedwith differences in hearing abilities associated to age-relatedhearing impairment (42). Likewise, pathogenic variants in ACP6have been recently identified in children with cerebral visualimpairment, which results from the impairment in projectionand/or interpretation of the visual input in the brain, and whichusually co-occurs with intellectual disability (43). The gene isexpressed at low levels in the brain (Figure 7).

Regarding CHD1L, this gene encodes a DNA helicaseprotein involved in the relaxation following DNA damage andultimately, in DNA repair, and its overexpression has beenlinked to several types of cancers (45). CHD1L is activatedby PARP1 during the initial steps of cellular reprogramming(46, 47). Interestingly, PARP1 is one of the genes that aredifferentially expressed among (mammalian) vocal learners (48),and its product regulates as well the dyslexia-susceptibility geneDYX1C1, important for neuronal migration in the developingcortex (49). We didn’t find evidence of a differential expressionof PARP1 in the blood of our patient compared to his mother,who does not exhibit speech or language problems, although

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FIGURE 3 | Chromosomal alterations and linguistic profile of the proband’s mother. (A) Screen capture of the array-CGH of the chromosome 1 of the proband’s

mother showing the microduplication at 1q21.1. (B) Developmental profile of the proband’s mother according to the verbal component of the WAIS-III (for

comparison, the figure includes the profile of the healthy, non-carrier father). The pink horizontal bar shows the normal values. (C) Reading abilities of the proband’s

mother according to the PROLEC-SE test (for comparison, the figure includes the profile of the healthy, non-carrier father: the low scores obtained by the father are

seemingly explained by his low educational level).

FIGURE 4 | DECIPHER patients of interest bearing chromosomal duplications at 1q21.1 with similar or smaller sizes than the one found in the proband. Patients with

cognitive deficits are highlighted in red, cases with no cognitive problems are highlighted in blue, whereas patients for whom no phenotypic profile is available are

highlighted in green.

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FIGURE 5 | Interaction network of the proteins coded for the most relevant genes duplicated in the proband. The network was drawn with String [version 10.5; (39)]

license-free software (http://string-db.org/), using the molecular action visualization. It includes the products of the protein-coding genes duplicated in the subject,

their potential interactors according to String, and the products of 15 strong candidates for language development/evolution. The proteins for which the expression

level of the gene was measured in the blood of the patient by qRT-PCR are framed in red (the product of the pseudogene HYDIN2 is not included in the network).

Colored nodes symbolize gene/proteins included in the query (small nodes are for proteins with unknown 3D structure, while large nodes are for those with known

structures). The color of the edges represent different kind of known protein-protein associations. Green: activation, red: inhibition, dark blue: binding, light blue:

phenotype, dark purple: catalysis, light purple: post-translational modification, black: reaction, yellow: transcriptional regulation. Edges ending in an arrow symbolize

positive effects, edges ending in a bar symbolize negative effects, whereas edges ending in a circle symbolize unspecified effects. Gray edges symbolize predicted

links based on literature search (co-mentioned in PubMed abstracts). Stronger associations between proteins are represented by thicker lines. The medium

confidence value was 0.0400 (a 40% probability that a predicted link exists between two enzymes in the same metabolic map in the KEGG database: http://www.

genome.jp/kegg/pathway.html). The diagram only represents the potential connectivity between the involved proteins, which has to be mapped onto particular

biochemical networks, signaling pathways, cellular properties, aspects of neuronal function, or cell-types of interest.

the gene is slightly upregulated in both carriers compared tothe healthy father (Supplementary Table 3). CHD1L is also acandidate for ASD and ADHD (25, 26). Contrary to what isobserved in patients with microdeletions of the 1q21.1 region,as the size of duplications encompassing CHD1L increases, ASDseverity also increases, whereas nonverbal IQ remains unaffected(25). Interestingly, DECIPHER patient 285509, who bears amicroduplication affecting only to part of the gene FMO5 and thewhole sequence of CHD1L, exhibits delayed speech and languagedevelopment (Figure 3). CHD1L is highly expressed in differentbrain areas, particularly in the cerebellum, where it is significantlyupregulated around birth (Figure 7). The cerebellum is involvedin almost every aspect of speech and language processing, fromsyntax processing to phonological and semantic verbal fluency,verbal working memory and speech perception and planning(50). Moreover, it is involved as well in most of the processes thatare impaired in ASD, from language and communication to socialinteraction and behavior, and cerebellar damage has been foundto contribute to the autistic phenotype (51).

We have found in the literature evidence of the potentialinvolvement of BCL9 in the language deficits observed in patientswith the 1q21.1 duplication syndrome. Similarly to CHD1L,BCL9 is also highly expressed in several parts of the brain,and particularly, in the cerebellum (Figure 7). BCL9 plays asignificant role in embryonic body axis development (52), aspart of the Wnt/β-catenin pathway (53). Specifically, BCL9promotes transcriptional activity and nuclear retention of β-catenin (54). β-catenin is encoded by CTNNB1, a gene relatedto cognitive disorders entailing language deficits, particularlyto schizophrenia (55, 56), but also to genes that have beenhypothesized to contribute to language evolution in our species[see (34) for details]. BCL9 itself is a candidate for schizophrenia(27) and interacts as well with genes important for languagedevelopment and evolution. To begin with, in mice, Bcl9 is atarget of Foxp2, the renowned ‘language gene’ (57). Additionally,BCL9 interacts with ARX to regulate canonical Wnt signaling(58). ARX is also a target of FOXP2 (57, 59) and regulatesthe migration of GABAergic interneurons (60), as well as

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FIGURE 6 | Expression profile of genes of interest in the blood of the proband. Only the genes showing significant (p < 0.05) differences with his healthy carrier

mother (A) and his healthy non-carrier father (B) are displayed. Variation in gene expression levels is expressed as fold changes.

dopaminergic neuron development (61). In humans, mutationsof ARX result in mental retardation and interneuronopathies(62), as well as in agenesis of the corpus callosum (63),a condition entailing deficits in problem solving and socialskills that often fall within the autistic spectrum (64, 65).Finally, according to the Reactome Pathway Database (https://reactome.org/), BCL9 interacts with both TLE2 and TLE3 aspart of the pathway involved in the activation and deactivationof the β-catenin transactivating complex. Both genes encodecorepressors of gene expression and play crucial functions inbrain development (66, 67). TLE3 is a target of FOXP2 inthe inferior prefrontal cortex (68). Interestingly, we have foundthat TL3 is significantly downregulated in the blood of ourproband compared to their asymptomatic parents. The gene isupregulated in the embryonic brain, particularly, in the neocortex(Figure 7).

Finally, among the set of robust candidates for languagedisorders and language evolution, we have found that ROBO1is strongly upregulated in the blood of our subject compared tohis healthy father and particularly, to his asymptomatic carriermother. ROBO1 encodes an axon guidance receptor involvedin interneuron migration and axon tract development in theforebrain, in particular, of thalamocortical axons implicated indiverse cognitive functions, consciousness, and alertness (69,70). Interestingly, ROBO1 regulates interaural interaction inauditory pathways (71), and was co-opted for vocalization insongbirds as a component of the motor song output nucleus (72).Overall, as part of the SLIT/ROBO pathway, ROBO1 has beenhypothesized to be a crucial candidate for speech developmentand evolution [see (34) for details]. The gene is a candidate forclinical conditions entailing speech problems, like dyslexia andspeech-sound disorder (73, 74). According to its known role in

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FIGURE 7 | Expression pattern in the body and the brain of genes of interest in the proband. (Left) Expression levels of the gene in different tissues according to the

Genotype-Tissue Expression (GTEx) project (44). Statistical analysis and data interpretation was performed by The GTEx Consortium Analysis Working Group. Data

was provided by the GTEx LDACC at The Broad Institute of MIT and Harvard. TPM, transcripts per million. (Right) Brain expression profile of the gene across

development. The expression data are from the Human Brain Transcriptome Database (http://hbatlas.org/). Six different brain regions are considered: the cerebellar

cortex (CBC), the mediodorsal nucleus of the thalamus (MD), the striatum (STR), the amygdala (AMY), the hippocampus (HIP), and 11 areas of neocortex (NCX).

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vocalization, the gene is highly expressed in several subcorticalregions (Figure 7). No DECIPHER patient bears a duplication ofROBO1 only, so it is difficult to ascertain the effects in humansof an excessive dosage of the gene. Nonetheless, it is interestingthat increased expression of Robo1 in rats reactivates astrocytesafter transient forebrain ischemia (75), whereas the increase ofRobo1 function in mice restores normal axon guidance in severalneuron populations (76).

In conclusion, although the exact molecular causes of thecognitive and linguistic profile of our proband remain to be fullyelucidated, we hypothesize that his distinctive features mightresult, to a large extent, from the overexpression of CHD1L.We further hypothesize that his problems with speech mightresult from subtle changes in the expression level of some ofthe genes involved in the externalization of language as part ofthe FOXP2/ROBO/SLIT regulatory networks, in particular, ofROBO1, but also some of the FOXP2’s targets, like TLE2. Wewish to acknowledge two potential caveats of our study. First,we have determined changes in the expression level of genes ofinterest in the blood, whereas the phenotype under scrutiny hasa brain-based locus of impairment. Nonetheless, as shown inFigure 7, our candidates are expressed in the blood and the brainat similar levels. Moreover, several studies point to a significantoverlapping in gene expression patterns between both tissues,ranging from 20% (77, 78) to 55% (79). Accordingly, we regardthat our findings in the blood can be confidently extrapolated tothe brain. Second, because the child was born following a multi-factorial high-risk pregnancy that involved maternal diabetesand exposure to anxiolytic drugs during embryonic and earlyfetal development, we cannot rule out the possibility that theseenvironmental influences contributed to his language problems.Such an effect is really difficult to estimate. Although recentresearch has acknowledged diabetes as a robust predictor oflanguage delay in twins (80), the available literature is not fullyconclusive [see (81) for a meta-review], and in fact, most childrenborn from mothers with diabetes are not significantly affected ifdiabetes is properly controlled during pregnancy [see (82) fordiscussion]. The effect of anxiolytics is perhaps more elusive.Considering the phenotype of our proband, it is interestingthat prenatal administration of diazepam delays the maturation

of temporal acuity in the rat auditory system (83). This effectmight result in altered speech perception in humans. However,no association has been found between anxiolytic exposureduring pregnancy and long-term language competence (84).Accordingly, considering as well all the evidence discussed inthe paper, we think that the language problems observed in ourproband are mostly due to the microduplication in chromosome1. We hope our findings contribute to a better understanding ofthe genetic underpinnings of human language.

AUTHOR CONTRIBUTIONS

AB-B conceived and planned the paper, analyzed the data,and wrote the manuscript. MB-M and IE-P performed thecytogenetic analyses and the microarrays, and analyzed the data.MF-U analyzed in detail the proband’s language in use speechand revised the manuscript. RT-R and SR-P performed the qRT-PCRs and analyzed the data. MJ-R assessed the cognitive andlinguistic problems of the child, analyzed the data, and revised themanuscript. All authors read and approved the final manuscript.

ACKNOWLEDGMENTS

We would like to thank the proband and his family for theirparticipation in this research. Preparation of this work wassupported by funds from the Spanish Ministry of Economyand Competitiveness (grant number FFI2016-78034-C2-2-P[AEI/FEDER,UE] to AB-B, with MJ-R, MB-M, and IE-P asmembers of the project).

SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be foundonline at: https://www.frontiersin.org/articles/10.3389/fped.2018.00163/full#supplementary-material

Supplementary Table 1 | List of primers used for qRT-PCR analyses.

Supplementary Table 2 | The whole set of genes important for language used

for the in silico analysis with String 10.5.

Supplementary Table 3 | Gene expression levels in the proband and his parents

in selected genes of interest.

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Frontiers in Pediatrics | www.frontiersin.org 15 June 2018 | Volume 6 | Article 163